Platelets are important in angiogenesis regulation and may influence the pathogenesis of ROP (4, 5). Thrombocytopenia is common in preterm infants (6, 7) and is frequently associated with sepsis and necrotizing enterocolitis (NEC) (8, 9). A case report suggests that platelet transfusion during proliferative ROP might inhibit neovascularization (NV) (10). Studies have shown an association between severe ROP and thrombocytopenia in neonatal life (10–14), but the influence of platelets in animal models of ROP has not been reported. In preterm infants born at gestational age (GA) <27 weeks (n = 202), we correlated ROP with platelet counts from birth to 36 PMA and found a correlation between thrombocytopenia in phase II (neovascular ROP) and the severity of ROP. To determine if platelet transfusion during this phase of ROP might suppress ROP development, we evaluated platelet counts in mice with oxygen-induced retinopathy (OIR) compared with controls and tested the effects of platelet transfusions (with and without granules) and platelet depletion on retinopathy.

Resting platelet, but not degranulated platelet transfusions, suppressed NV. To determine whether the observed effects of platelets were dependent upon the release of their granule content, we generated degranulated platelets with no α-granules by exposing washed platelets to thrombin (15–17). P-selectin is stored in secretory granules of platelets and is rapidly expressed on the surface upon activation. The expression of P-selectin, therefore, reflects the degranulation of platelets (15). Activation with 1U/ml thrombin led to P-selectin expression in 93% of platelets (versus 0.7% of unstimulated resting platelets), indicating degranulation of most platelets (Figure 5, A and B). To confirm comparable clearance rates, degranulated and resting GFP+ platelets were followed over time after transfusion in neonatal P17 pups. No significant difference in clearance was seen between groups (Figure 5C). In the OIR model, NV was significantly reduced in the resting platelet transfusion group compared with the degranulated platelet transfusion group (P = 0.039). VO was unchanged (Figure 5D).

We investigated longitudinal platelet counts in relation to the development of severe ROP clinically and experimentally (in a mouse model of ROP). We then evaluated the effects of platelet transfusions and depletion on proliferative ROP in OIR in mice. The most prominent factor associated with severe ROP in our retrospective analysis was any episode of thrombocytopenia at ≥30 weeks PMA, even after adjusting for well-known confounding factors such as GA, NEC, and sepsis. This is in agreement with 2 case-control studies and a longitudinal study reporting that thrombocytopenia was independently associated with severe ROP requiring treatment (11, 12, 14). In our study, all infants with severe ROP requiring laser treatment with any thrombocytopenia at ≥30 weeks PMA also had some thrombocytopenia in the initial vessel loss phase of ROP (<30 weeks PMA), indicating a possible multi-hit pattern (13).

In the oxygen-induced retinopathy mouse model, the reduction of vasoproliferation at the beginning of neovascular tuft formation (P15–P16) after platelet transfusions was in agreement with a report of regression of severe ROP in human preterm neonates after platelet transfusions during proliferative retinopathy (10). The increased NV after platelet depletion supports the hypothesis and is in line with the clinical correlation seen in our retrospective analysis.

The effect of VEGFA in the oxygen induced retinopathy model (OIR) has been intensively investigated and was identified as the most important mediator of pathological NV in the disease. In phase I of the ROP model of OIR, high oxygen levels suppress retinal VEGFA expression, which leads to central VO. Upon return to room air at P12, hypoxia in the central avascular area leads to upregulation of VEGFA, primarily by Müller cells and astrocytes (18). The rapid increase in retinal VEGFA leads to NV into the vitreous starting at P14–P15. In our murine study, platelet depletion led to an increase, and platelet transfusion to a decrease, in retinal Vegfa mRNA and VEGFA protein expression. The exact mechanism of how platelets in the bloodstream influence retinal VEGFA expression is unclear. However, Müller glia cells (where most hypoxia-induced VEGFA is expressed in OIR) have close interactions with both vessels and neurons, and they play an important role in the homeostasis of the retina (19). It is therefore possible that an antiangiogenic stimulus released from platelets is communicated through endothelial cells to Müller cells, resulting in downregulation of Vegfa mRNA and VEGFA protein expression.

Platelets have numerous bioactive molecules stored in 3 types of secretory granules including α-granules, dense granules, and lysosomes, enabling them to exert a wide range of effects on the surrounding microenvironment (20). α-Granules are the most abundant and contain over 100 bioactive proteins including various pro- and antiangiogenic factors such as angiostatin, endostatin, platelet factor-4, thrombospondin-1, α2-macroglobulin, and plasminogen activator inhibitor 1 (21, 22). Even though platelets contain pro- and antiangiogenic factors, the reported effect of platelets on endothelial cells has mainly been proangiogenic or proproliferative (23–27). Pipili-Synetos et al. (23) showed a proliferative effect of platelets on HUVEC in vitro for the first time in 1998. Enhanced endothelial cell tube formation was seen in a dose-dependent manner with stimulation with resting platelets but, interestingly, not with α-thrombin–stimulated platelet supernatant. Pipili-Synetos and colleagues (23) concluded that a direct interaction between endothelium and platelets is necessary to promote the proproliferative effect. Further, data from clinical and experimental cancer research also supports a stimulatory effect of platelet on tumor angiogenesis (28). Nevertheless, the observation by Italiano et al. that pro- and antiangiogenic proteins are separated in specific subpopulations of α-granules inspired the hypothesis that platelets can locally promote or inhibit angiogenesis through controlled granule release (5). A follow-up investigation by the same group showed that angiogenic proteins can be differentially secreted in different in vitro conditions. Stimulation of washed platelets with the platelet activators ADP and Thromboxane A2 (TXA2) led to a differential release of VEGF (stimulant of vessel growth) or endostatin (inhibitor of vessel growth), respectively. In accord, a divergent angiogenic effect was seen in a tube formation assay after stimulation with platelet releasate generated by ADP and TXA2, demonstrating that platelets can stimulate or suppress angiogenesis in vitro, depending on conditions (29). The in vitro data is further supported by Ma et al. studies investigating the effect of platelets on gastric ulcer healing in rats in vivo (27). Treatment with the ADP antagonist Ticlopidine increased serum endostatin and decreased VEGF levels, which resulted in decreased gastric ulcer healing. In the same study, serum from Ticlopidine-treated rats reduced angiogenesis in a sprouting assay in vitro (27).

Our murine study is, to our knowledge, the first study showing a reduction of NV in vivo by platelets. We hypothesize that the unique microenvironment in the pathological neovascular tuft area at P15–P17 leads to a controlled release of antiangiogenic factors, resulting in the suppression of neovascular tuft formation and downregulation of retinal VEGFA expression through neurovascular crosstalk. Platelet transfusion or platelet depletion did not affect the area of VO in the retina in the OIR model.

In order to distinguish whether platelet granule release or the platelet interaction with endothelium by itself causes the suppression of NV, we generated degranulated platelets by activating washed platelets in vitro with thrombin. We compared the effect of degranulated versus resting platelets with intact granules and found that degranulated platelets, unlike resting platelets, did not affect NV. This supports the hypothesis that factors within the granules are important to suppress NV.

In order to more fully address the mechanism with respect to which platelet factors mediate the protective effect on ROP, we need mice with single or possibly multiple genetic depletions of factors specifically in platelets. These mice need to be generated, which will take considerable time and will be part of future investigations in our laboratories.

Overall, our findings suggest a local interactive effect of platelets specifically on endothelial cells in neovascular tufts that leads to direct inhibition of NV, as well as a secondary regulation of VEGFA expression in the neural retina, ultimately controlling NV in ROP. Our data suggest that platelets are important for normal fetal/neonatal vascular development and that, during the proliferative phase of ROP, low platelet counts are detrimental, likely due to a lack of platelet factors as yet to be determined. The risks and benefits of platelet transfusions in preterm infants, a population with unique physiology and pathological processes, are in general poorly understood (6, 30), and transfusions should be carefully considered.

In summary, clinical thrombocytopenia at PMA >30 weeks (at the onset of proliferative retinopathy) is a prominent factor associated with severe ROP requiring treatment. In addition, in the mouse model of ROP at the onset of neovascular tuft formation, platelet depletion increases retinopathy while platelet transfusion decreases retinopathy.

Patients. We retrospectively reviewed 237 infants born at GA <27 weeks and screened for ROP in Stockholm from 2008–2011 (n = 176) and Gothenburg from 2013–2015 (n = 61). International guidelines for ROP classification and ROP treatment were followed (31, 32). We retrieved ROP data from the Swedish national register for ROP (SWEDROP) (33). We defined the initial phase of ROP, with suppressed vascularization, as <30 weeks PMA and the later proliferative phase as 30–36 weeks PMA. Sepsis was defined as a positive blood culture and/or clinical signs and C-reactive protein >20 mg/l. Platelet counts and platelet transfusions from birth until 36 weeks PMA were recorded; 35 infants were excluded for lack of platelet counts until at least 30 weeks PMA. Thrombocytopenia was defined as any platelet count <100 × 109/l. Mean platelet count per day was assessed for each infant based on these data, and a mean weekly platelet count was calculated for each infant.

OIR. The mouse OIR model was used as described (34–36). Briefly, C57BL/6 mouse pups (with nursing dams) (The Jackson Laboratory) are exposed to 75% oxygen from P7–P12 to induce retinal vessel loss and returned to room air at P12–P17, when hypoxic retina stimulates NV. Mice with body weight under 5.5 g or over 7.8 g were excluded in this study. Maximum retinal NV and remaining VO at P17 was quantified (Figure 2A).

Platelet degranulation. For degranulation, platelet suspensions were stimulated for 10 minutes with 1 U/ml thrombin (Chrono-log) under stirring conditions at 37°C in a Chrono-log platelet aggregometer in the presence of 2 mM EDTA (IBI Scientific) to avoid aggregation. The reaction was stopped by the addition of hirudin (Pentapharm), as previously described (15). Resting platelet suspensions were treated in the same way, with the exception that an equal volume of Tyrode’s buffer was added instead of thrombin. Platelet suspensions were spun down at 700 g, the supernatant was removed, and platelet pellets were resuspended in sterile saline to a concentration of 2.6 × 106 to 3.3 × 106 platelets/μl and left for 30 minutes at 37°C before use. Platelet degranulation was confirmed by flow cytometry analysis of P-selectin expression on the platelet surface. Briefly, 20,000 platelet/μl in a final volume of 40 μl were stained for 15 minutes at RT with an AF647-coupled anti–P-selectin antibody (BD Pharmingen, catalog 563674) and a PE-coupled anti-CD41 antibody (BD Pharmingen, catalog 558040). The reaction was stopped by the addition of 300 μl PBS and data obtained on a FACS Canto (BD Biosciences). Comparable platelet lifespan rates in circulation between platelet preparations were ensured by measuring the percentage of GFP+ degranulated and resting platelets in whole blood after transfusion in WT mice through FACS analysis. Recipient mice were transfused with 10 μl per gram body weight of degranulated or resting platelets through retro-orbital injection on days P15 and P16.

Platelet depletion in mouse pups. To deplete platelets in mice with OIR, 2 μg per gram body weight anti-GPIbα (CD42b) antibody (R300, Emfret) in sterile PBS was injected i.p. in P15 mice. Littermates were injected with PBS as controls. The phenotype experiment was also repeated with IgG as control. A comparable effect was seen with IgG or PBS as control (Supplemental Figure 2.)

Protein and RNA analyses. Following enucleation, retinas were immediately dissected, washed in PBS, and cleaned from the remaining vitreous. The retina was cut in half for RNA and protein analyses. Total RNA was extracted from mouse retina using RNeasy kit (Qiagen) and reverse-transcribed with SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). A SYBR-green based real-time PCR was used for quantification. VEGFA plasma and retina protein levels were quantified using standard mouse VEGF ELISA Kit (Quantikine ELISA, R&D Systems). Briefly, retinal homogenized in PBS by sonication, centrifuged for 5 minutes at 5,000 g and assayed according to standard protocol. Blood was collected after enucleation, transferred to an EDTA-coated tube, and centrifuged for 20 minutes at 2,000 g. Plasma supernatant was diluted 1:5 and assayed according to standard protocol. A microplate reader set at 450 nm with wavelength correction for 540 nm was used for optical density measurement.

Animals. WT C57BL/6 mice from the Jackson Laboratory were used for all platelet transfusion and depletion experiments. For proof-of-concept experiments, 129S6 mice from Taconic were included in the analysis. GFP+ platelets were isolated from adult C57BL/6Tg(UBC-GFP)30Scha/J mice (strain no. 004353; the Jackson Laboratory). Both sexes were included in this study.

Statistics. Statistical analysis was performed using GraphPad Prism 6 and IBM SPSS Statistics 24 for Microsoft Windows (IBM). For the preclinical study, a 2-tailed unpaired t test was used for comparison between 2 groups. In the clinical data analysis χ2 test and Mann-Whitney U test was used for comparison between 2 groups. OR and adjusted OR were calculated using logistic regression analysis. A P value below or equal 0.05 was considered as significant.

Study approval. The clinical study was approved by the hospital review boards and carried out in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All animal studies adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Boston Children’s Hospital IACUC. International guidelines for ROP classification and ROP treatment were followed (31, 32).

PL, RL, BC, and GH contributed equally to this work. PL, GH, and AH designed and performed the clinical research, collected and analyzed data, and wrote the manuscript; BC, RL, YS, ZJL, MCSV, and LEHS designed and performed the preclinical research, collected and analyzed data, and wrote the manuscript. SC, AP, RD, VL, and JC collected and analyzed data. SK, GH, CM, CL, and BH provided advice, assisted with data collection and statistical analyzes, and approved the manuscript.

Gilbert C, et al. Characteristics of infants with severe retinopathy of prematurity in countries with low, moderate, and high levels of development: implications for screening programs. Pediatrics. 2005;115(5):e518–e525.

Early Treatment For Retinopathy Of Prematurity Cooperative Group. Revised indications for the treatment of retinopathy of prematurity: results of the early treatment for retinopathy of prematurity randomized trial. Arch Ophthalmol. 2003;121(12):1684–1694.